Phosphoproteomic analysis reveals distinctive responses in Mangrovibacter phragmatis under high-salinity condition.

We conducted a thorough genome-wide investigation of protein phosphorylation in the halotolerant bacterium Mangrovibacter phragmitis (MPH) ASIOC01, using the Fe-IMAC enrichment method combined with tandem mass spectrometry under low- and high-salinity conditions. The phosphoproteome comprises 86 unique phosphorylated proteins, crucially involving pathways such as glycolysis/gluconeogenesis, the citrate cycle, chaperones, ribosomal proteins, and cell division. This study represents the first and most extensive investigation to-date comparing the bacterial phosphoproteome under different osmotic conditions using a gel-free approach. We identified 45 unique phosphoproteins in MPH cultured in media containing 1 % NaCl, and 33 exclusive phosphoproteins in MPH cultured in media containing 5 % NaCl. Eight phosphoproteins were detected in both growth conditions. Analysis of high-confidence phosphosites reveals that phosphorylation predominantly occurs on serine residues (52.3 %), followed by threonine (35.1 %) and tyrosine (12.6 %) residues. Interestingly, 34 % of the phosphopeptides display multiple phosphosites. Currently, prokaryotic phosphorylation site prediction platforms like MPSite and NetPhosBac 1.0 demonstrate an average prediction accuracy of only 21 % when applied to our dataset. Fourteen phosphoproteins did not yield matches when compared against dbPSP 2.0 (database of Phosphorylation Sites in Prokaryotes), indicating that these proteins may be novel phosphoproteins. These unique proteins undergoing phosphorylation under high salinity growth conditions potentially enhance their adaptive capabilities to environmental challenges.

Experimental evolution of extremotolerant and extremophilic fungi under osmotic stress.

Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the halotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale were grown at high NaCl or glycerol concentrations, and the halophilic basidiomycete Wallemia ichthyophaga was grown close to its lower NaCl growth limit. All evolved Aureobasidium spp. accelerated their growth at low water activity. Whole genomes of the evolved strains were sequenced. No aneuploidies were detected in any of the genomes, contrary to previous studies on experimental evolution at high salinity with other species. However, several hundred single-nucleotide polymorphisms were identified compared with the genomes of the progenitor strains. Two functional groups of genes were overrepresented among the genes presumably affected by single-nucleotide polymorphisms: voltage-gated potassium channels in A. pullulans at high NaCl concentration, and hydrophobins in W. ichthyophaga at low NaCl concentration. Both groups of genes were previously associated with adaptation to high salinity. Finally, most evolved Aureobasidium spp. strains were found to have increased intracellular and decreased extracellular glycerol concentrations at high salinity, suggesting that the strains have optimised their management of glycerol, their most important compatible solute. Experimental evolution therefore not only confirmed the role of potassium transport, glycerol management, and cell wall in survival at low water activity, but also demonstrated that fungi from extreme environments can further improve their growth rates under constant extreme conditions in a relatively short time and without large scale genomic rearrangements.

Characterization of susceptibility patterns and adaptability of the newly emerged Candida auris.

The emergence of Candida auris has caused a major concern in the public health worldwide. This novel fungus is characterized by its multidrug resistance profile, ability to thrive in harsh and stressful conditions, as well as high temperatures and salt concentrations, persistence on hospital surfaces, causing nosocomial infections and outbreaks, and unique fitness properties. Here, we study the antifungal susceptibility patterns, thermotolerance, and halotolerance of 15 putative C. auris clinical isolates from Inkosi Albert Academic Hospital, Durban, South Africa. Five of the C. auris isolates showed resistance to all three antifungals (fluconazole, amphotericin B, and micafungin) and were selected for characterization of their adaptability mechanisms. Four of the tested multidrug-resistant C. auris isolates (C. auris strain F25, C. auris strain F276, C. auris F283, and C. auris M153) showed good growth when exposed to high temperature (42 °C) and salinity (10% NaCl) conditions whereas one isolate (C. auris F65) showed moderate growth under these conditions. Candida parapsilosis showed poor growth whereas C. albicans no growth under these conditions. The five C. auris strains were positive for all the adaptive features.

200 years of taxonomic confusion: Sporendonema and allies.

The genus Sporendonema (Gymnoascaceae, Onygenales) was introduced in 1827 with the type species S. casei for a red mould on cheese. Cheese is a consistent niche for this species. Sphaerosporium equinum is another species classified in Gymnoascaceae and has also been reported from cheese. Recently, other habitats have been reported for both Sporendonema casei and Sphaerosporium equinum. The present study aimed to investigate the taxonomy of Sporendonema and Sphaerosporium, as well as a close neighbour, Arachniotus. Two strains of Hormiscium aurantiacum, another related cheese-associated species were also included in the analyses. Strains were evaluated in terms of macro- and micromorphology, physiology including salt tolerance, growth rate at different temperatures, casein degradation, cellulase activity, lipolytic activity, and multi-locus phylogeny with sequences of the nuclear ribosomal internal transcribed spacer region, the D1-D2 region of the large subunit and partial ß-tubulin locus sequences. The results showed that the analysed species were congeneric, and the generic names Arachniotus and Sphaerosporium should be reduced to the synonymy of Sporendonema. Therefore, four new combinations as well as one lectotype and one epitype were designated in Sporendonema. Two strains attributed to Sphaerosporium equinum from substrates other than cheese were found to be phylogenetically and morphologically deviant and were introduced as a new species named Sporendonema isthmoides.

Halotolerance mechanisms in salt­tolerant cyanobacteria.

Cyanobacteria are ubiquitously distributed in nature and are the most abundant photoautotrophs on Earth. Their long evolutionary history reveals that cyanobacteria have a remarkable capacity and strong adaptive tendencies to thrive in a variety of conditions. Thus, they can survive successfully, especially in harsh environmental conditions such as salty environments, high radiation, or extreme temperatures. Among others, salt stress because of excessive salt accumulation in salty environments, is the most common abiotic stress in nature and hampers agricultural growth and productivity worldwide. These detrimental effects point to the importance of understanding the molecular mechanisms underlying the salt stress response. While it is generally accepted that the stress response mechanism is a complex network, fewer efforts have been made to represent it as a network. Substantial evidence revealed that salt-tolerant cyanobacteria have evolved genomic specific mechanisms and high adaptability in response to environmental changes. For example, extended gene families and/or clusters of genes encoding proteins involved in the adaptation to high salinity have been collectively reported. This chapter focuses on recent advances and provides an overview of the molecular basis of halotolerance mechanisms in salt­tolerant cyanobacteria as well as multiple regulatory pathways. We elaborate on the major protective mechanisms, molecular mechanisms associated with halotolerance, and the global transcriptional landscape to provide a gateway to uncover gene regulation principles. Both knowledge and omics approaches are utilized in this chapter to decipher the mechanistic insights into halotolerance. Collectively, this chapter would have a profound impact on providing a comprehensive understanding of halotolerance in salt­tolerant cyanobacteria.

Unravelling the potential plant growth activity of halotolerant Bacillus licheniformis NJ04 isolated from soil and its possible use as a green bioinoculant on Solanum lycopersicum L.

Immensely expanding world population and narrowing arable land for agriculture is a mighty concern faced by the planet at present. One of the major reasons for decline in arable lands is the increased soil salinity, making it unfavourable for crop cultivation. Utilisation of these saline land for agriculture is possible with suitable invention for improving the soil quality. Biofertizers manufactured out of Plant Growth Promoting Rhizobacteria is one such innovation. In the present study, Bacillus licheniformis NJ04 strain was isolated and studied for its halotolerance and other effective plant growth promoting traits. The NJ04 strain was able to tolerate salt up to 10% and highlighted remarkable antifungal activity against common fungal phytopathogens. The preliminary seed germination test in Solanum lycopersicum seeds revealed a significant increase in root length (16.29 ± 0.91 cm) and shoot length (9.66 ± 0.11 cm) of treated plants as compared with the control plants and thereby shows its possible use as a green bioinoculant in agriculture and an ideal candidate to compete with salt stress.

Halotolerance, stress mechanisms, and circadian clock of salt-tolerant cyanobacteria.

Cyanobacteria harbor a high level of physiological flexibility, which enables them to reside in virtually all available environmental niches, including extreme environments. In this review, we summarize the recent advancements in stress mechanisms of salt-tolerant (a.k.a. halotolerant) cyanobacteria. Omics approaches have been extensively employed in recent years to decipher mechanisms of halotolerance and to understand the relevance of halotolerance-associated gene regulatory networks. The vast knowledge from genome mining disclosed that halotolerant cyanobacteria possess extended gene families and/or clusters, encoding enzymes that synthesize unique osmoprotectants, including glycine betaine (GB), betaine derivatives, and mycosporine-like amino acids (MAAs). Comprehensive transcriptomic analyses were conducted using Halothece sp. PCC7418 (hereafter referred to as Halothece), a cyanobacterium that exhibits remarkable halotolerance. These studies revealed a specific transcriptional response when Halothece was subjected to salt stress, whereas salt and osmotic stresses were found to share a common transcriptomic response. Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. Lastly, novel insights highlight the relationship between the molecular regulation of the circadian rhythm and salt stress tolerance. Since the circadian rhythm of gene expression was distorted under salt stress, halotolerant cyanobacteria may prioritize the adaptation to salt stress by attenuation of circadian rhythmicity. KEY POINTS: • Recent advancements in the understanding of stress mechanisms in halotolerant cyanobacteria are described based on omics analyses. • Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. • Since salt stress affects the molecular regulation among clock-related proteins, salt stress may attenuate circadian rhythmicity.

Decolorization of reactive azo dye using novel halotolerant yeast consortium HYC and proposed degradation pathway.

The presence of high salinity levels in textile wastewater poses a significant obstacle to the process of decolorizing azo dyes. The present study involved the construction of a yeast consortium HYC, which is halotolerant and was recently isolated from wood-feeding termites. The consortium HYC was mainly comprised of Sterigmatomyces halophilus SSA-1575 and Meyerozyma guilliermondii SSA-1547. The developed consortium demonstrated a decolourization efficiency of 96.1% when exposed to a concentration of 50 mg/l of Reactive Black 5 (RB5). The HYC consortium significantly decolorized RB5 up to concentrations of 400 mg/l and in the presence of NaCl up to 50 g/l. The effects of physicochemical factors and the degradation pathway were systematically investigated. The optimal pH, salinity, temperature, and initial dye concentration were 7.0, 3%, 35 °C and 50 mg/l, respectively. The co-carbon source was found to be essential, and the addition of glucose resulted in a 93% decolorization of 50 mg/l RB5. The enzymatic activity of various oxido-reductases was assessed, revealing that NADH-DCIP reductase and azo reductase exhibited greater activity in comparison to other enzymes. UV-Visible (UV-vis) spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) were utilized to identify the metabolites generated during the degradation of RB5. Subsequently, a metabolic pathway was proposed. The confirmation of degradation was established through alterations in the functional groups and modifications in molecular weight. The findings indicate that this halotolerant yeast consortium exhibits promising potential of degrading dye compounds. The results of this study offer significant theoretical basis and crucial perspectives for the implementation of halotolerant yeast consortia in the bioremediation of textile and hypersaline wastewater. This approach is particularly noteworthy as it does not produce aromatic amines.

Dynamics of the role of LacMeta laccase in the complete degradation and detoxification of malachite green.

Laccases highlight for xenobiotic bioremediation, as well as application in the fine chemical, textile, biofuel and food industries. In a previous work, we described the preliminary characterization of laccase LacMeta, a promising enzyme for the bioremediation of dyes, able to decolorization malachite green (MG), trypan blue, methylene blue. Here we demonstrate that LacMeta is indeed suitable for the complete degradation and detoxification of MG dye, not just for its discoloration, since some works show false positives due to the formation of colorless intermediates such as leucomalachite. The optimal pH and temperature parameters of LacMeta were 5.0 and 50 °C, respectively (MG as substrate). LacMeta was tolerant of up to 10 mmol L- 1 EDTA (82%) and up to 5% (V/V) acetone (91%) and methanol (71%), while SDS promoted severe inhibition. For ions, a high tolerance to cobalt, zinc, manganese, and calcium (10 mmol L- 1) was also observed (> 90%). Even under high-salinity conditions (1 mol L- 1 NaCl), the residual bleaching activity of the dye remained at 61%. Furthermore, the bleaching product of MG did not inhibit the germination of sorghum and tomato seeds and was inert to the vegetative structures of the germinated seedlings. Additionally, this treatment effectively reduced the cytotoxic effect of the dye on microorganisms (Escherichia coli and Azospirillum brasilense), which can be explained by H-NMR spectral analysis results since LacMeta completely degraded the peak signals corresponding to the aromatic rings in the dye, demonstrating extreme efficiency in the bioremediation of the xenobiotic at high concentrations (50 mg L- 1).

High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) ß-mannanase from Bacillus velezensis H1.

ß-mannanase catalyzes the hydrolysis of mannans ß-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant ß-mannanases are already reported, existence of a high halotolerant ß-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant ß-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The Km and Vmax values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.

Maturation Process and Characterization of a Novel Thermostable and Halotolerant Subtilisin-Like Protease with High Collagenolytic Activity but Low Gelatinolytic Activity.

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a ß-jelly roll (ßJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the ßJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca2+-binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1' positions, respectively. Both the ßJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Differential analysis of ergosterol function in response to high salt and sugar stress in Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii is an osmotolerant and halotolerant yeast that can participate in fermentation. To understand the mechanisms of salt and sugar tolerance, the transcription levels of Z. rouxii M 2013310 under 180 g/L NaCl stress and 600 g/L glucose stress were measured. The transcriptome analysis showed that 2227 differentially expressed genes (DEGs) were identified under 180 g/L NaCl stress, 1530 DEGs were identified under 600 g/L glucose stress, and 1278 DEGs were identified under both stress conditions. Then, KEGG enrichment analyses of these genes indicated that 53.3% of the upregulated genes were involved in the ergosterol synthesis pathway. Subsequently, quantitative PCR was used to verify the results, which showed that the genes of the ergosterol synthesis pathway were significantly upregulated under 180 g/L NaCl stress. Finally, further quantitative testing of ergosterol and spotting assays revealed that Z. rouxii M 2013310 increased the amount of ergosterol in response to high salt stress. These results highlighted the functional differences in ergosterol under sugar stress and salt stress, which contributes to our understanding of the tolerance mechanisms of salt and sugar in Z. rouxii.

Pre-incubation conditions determine the fermentation pattern and microbial community structure in fermenters at mild hydrostatic pressure.

Fermentation at elevated hydrostatic pressure is a novel strategy targeting product selectivity. However, the role of inoculum history and cross-resistance, that is, acquired tolerance from incubation under distinctive environmental stress, remains unclear in high-pressure operation. In our here presented work, we studied fermentation and microbial community responses of halotolerant marine sediment inoculum (MSI) and anaerobic digester inoculum (ADI), pre-incubated in serum bottles at different temperatures and subsequently exposed to mild hydrostatic pressure (MHP; < 10 MPa) in stainless steel reactors. Results showed that MHP effects on microbial growth, activity, and community structure were strongly temperature-dependent. At moderate temperature (20°C), biomass yield and fermentation were not limited by MHP; suggesting a cross-resistance effect from incubation temperature and halotolerance. Low temperatures (10°C) and MHP imposed kinetic and bioenergetic limitations, constraining growth and product formation. Fermentation remained favorable in MSI at 28°C and ADI at 37°C, despite reduced biomass yield resulting from maintenance and decay proportionally increasing with temperature. Microbial community structure was modified by temperature during the enrichment, and slight differences observed after MHP-exposure did not compromise functionality. Results showed that the relation incubation temperature-halotolerance proved to be a modifier of microbial responses to MHP and could be potentially exploited in fermentations to modulate product/biomass ratio.

Production of a halotolerant endo-1,4-ß-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-ß-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-ß-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley ß-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-ß-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley ß-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-ß-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.

Global transcriptome analyses and regulatory mechanisms in Halothece sp. PCC 7418 exposed to abiotic stresses.

Halotolerant species are of interest since they occur naturally in environments with excess toxic ions. The cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece) exhibits remarkable halotolerance and was used to examine stress-responsive regulatory mechanisms. The effects of five different stimuli on Halothece transcriptomes were examined using RNA sequencing. In response to diverse stresses, there were both common and stress-specific transcriptional responses. A common upregulated gene set under all stresses consisted of nine differentially expressed genes (DEGs). We also found that osmotic stress elicited the largest set of DEGs. Salt- and osmotic-responsive regulatory mechanisms shared common pathways. DEGs that were upregulated under salt stress encoded proteins involved in photosynthesis and related machineries. Furthermore, DEGs encoding two-component system (TCS) factors, transcriptional factors, scaffolds for protein-protein interactions, transporters, protein turnover factors, and lipid biosynthesis enzymes were also identified under salt stress. Notably, one-carbon (1C) metabolism factors, glycine betaine (GB) synthesis enzymes, and GB transporters were upregulated under salt stress. Metabolic analyses revealed that GB accumulated under salt stress, while mycosporine-2-glycine (M2G) accumulated under salt or osmotic stress. None of the nutrient starvations induced GB nor M2G accumulation. These results suggested that GB and M2G are two osmoprotectants that contribute to halotolerance. Based on our results, we proposed regulatory mechanisms that are crucial for halotolerance, which are coordinated with the GB, M2G, 1C, amino acid, and central carbon interlinking metabolic pathways. 1C metabolism directly fulfills the high metabolite requirements for halotolerance together with the ancillary role of several metabolic pathways.Key Points• Global transcriptome surveys together with molecular and metabolite analyses provide insights into regulatory networks that are crucial for halotolerance• Regulatory networks that are crucial for halotolerance coordinated with the two key osmoprotectants, one carbon, amino acid, and central carbon interlinking metabolic pathways• The findings have translational relevance in genomic and transcriptomic mechanisms of halotolerance.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Recent developments in the biology and biotechnological applications of halotolerant yeasts.

Natural hypersaline environments are inhabited by an abundance of prokaryotic and eukaryotic microorganisms capable of thriving under extreme saline conditions. Yeasts represent a substantial fraction of halotolerant eukaryotic microbiomes and are frequently isolated as food contaminants and from solar salterns. During the last years, a handful of new species has been discovered in moderate saline environments, including estuarine and deep-sea waters. Although Saccharomyces cerevisiae is considered the primary osmoadaptation model system for studies of hyperosmotic stress conditions, our increasing understanding of the physiology and molecular biology of halotolerant yeasts provides new insights into their distinct metabolic traits and provides novel and innovative opportunities for genome mining of biotechnologically relevant genes. Yeast species such as Debaryomyces hansenii, Zygosaccharomyces rouxii, Hortaea werneckii and Wallemia ichthyophaga show unique properties, which make them attractive for biotechnological applications. Select halotolerant yeasts are used in food processing and contribute to aromas and taste, while certain gene clusters are used in second generation biofuel production. Finally, both pharmaceutical and chemical industries benefit from applications of halotolerant yeasts as biocatalysts. This comprehensive review summarizes the most recent findings related to the biology of industrially-important halotolerant yeasts and provides a detailed and up-to-date description of modern halotolerant yeast-based biotechnological applications.

Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation.

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

Hydrostatic pressure- and halotolerance of Photobacterium phosphoreum and P. carnosum isolated from spoiled meat and salmon.

Photobacterium spp. occur frequently in marine environments but have been recently also found as common spoilers on chilled meats. The environmental conditions in these ecological niches differ especially regarding salinity and ambient pressure. Linking the occurrence of photobacteria in different niches may elucidate its ecology and bring insights for the food industry. We investigated tolerance of Photobacterium (P.) phosphoreum and P. carnosum strains to high hydrostatic pressure and salinity and aligned our observations with presence of relevant genes. The strains were isolated from packaged meats and salmon (or the sea) to identify adaptations to marine and terrestrial habitats. Growth of all P. carnosum strains was reduced by 40 MPa hydrostatic pressure and >3% sodium chloride, suggesting loss of traits associated with marine habitats. In contrast, P. phosphoreum strains were only slightly affected, suggesting general adaptation to marine habitats. In accordance, these strains had gene clusters associated with marine niches, e.g. flagellar and lux-operons, being incomplete in P. carnosum. Occurrence of P. carnosum strains on packaged salmon and P. phosphoreum strains on meats therefore likely results from cross-contamination in meat and fish processing. Still, these strains showed intermediate traits regarding pressure- and halotolerance, suggesting developing adaptation to their respective environment.

Traversing the "Omic" landscape of microbial halotolerance for key molecular processes and new insights.

Post-2005, the biology of the salt afflicted habitats is predominantly studied employing high throughput "Omic" approaches comprising metagenomics, transcriptomics, metatranscriptomics, metabolomics, and proteomics. Such "Omic-based" studies have deciphered the unfamiliar details about microbial salt-stress biology. The MAGs (Metagenome-assembled genomes) of uncultured halophilic microbial lineages such as Nanohaloarchaea and haloalkaliphilic members within CPR (Candidate Phyla Radiation) have been reconstructed from diverse hypersaline habitats. The study of MAGs of such uncultured halophilic microbial lineages has unveiled the genomic basis of salt stress tolerance in "yet to culture" microbial lineages. Furthermore, functional metagenomic approaches have been used to decipher the novel genes from uncultured microbes and their possible role in microbial salt-stress tolerance. The present review focuses on the new insights into microbial salt-stress biology gained through different "Omic" approaches. This review also summarizes the key molecular processes that underlie microbial salt-stress response, and their role in microbial salt-stress tolerance has been confirmed at more than one "Omic" levels.